Oxidase systems have been exploited for determination of various substrates for many years. For example, in the presence of oxygen the enzyme glucose oxidase catalyzes the oxidation of glucose by oxygen to gluconolactone (gluconic acid) and hydrogen peroxide; lactate is oxidized by lactate oxidase; glutamic acid is oxidized by glutamate oxidase, etc; hydrogen peroxide being the by-product of all these reactions.
The concentration of the substrate, e.g. glucose, can be followed by monitoring the concentration of H.sub.2 O.sub.2. For example, addition of enzyme peroxidase and a chromogenic oxygen acceptor, such as colorless o-dianisidine, results in the reaction of hydrogen peroxide and o-dianisidine with formation of a measurable color (oxidized o-dianisidine, brown, absorption at 500 nm). The method is not widely used because O-dianisidine is toxic. In any case, this approach requires more than one enzyme (oxidase and peroxidase). Alternatively, H.sub.2 O.sub.2 can be monitored by NADH peroxidase and the expensive cofactor, NADH (a reduced form of nicotinamide adenine dinucleotide).
It is desirable to eliminate the use of peroxidase. Also, the spectrophotometric determination of glucose in some body fluids or solutions with pronounced color faces serious difficulty because of color masking. Blue dyes are advantageous in this regard because their absorption peaks are distant from the bands characteristic of most biological fluids.
Water-insoluble ferrocene [bis(.eta..sup.5 -cyclopentadienyl)iron] and its derivatives have been studied by Cass, A. E. G. et al, Anal. Chem. 56, 667-671 (1984) and by Hill, H. A. O. et al, Biosensors, a Practical Approach (Cass, A. E. G., Ed.), pp. 19-46, IRL Press, Oxford (1990) to develop mediated amperometric biosensors because of their electron-exchange properties with various enzymes. To replace oxygen as an electron receptor, ferrocene must be first oxidized at a platinum or carbon electrode surface to ferricinium through the loss of one electron from the metal atom.
The ferricinium cation is water-soluble and able to react with several reduced oxidoreductases as follows: EQU Substrate+Enzyme.sub.ox .fwdarw.Enzyme.sub.red +Product (1) EQU 2 Ferricinium+Enzyme.sub.red .fwdarw.2 Ferrocene+Enzyme.sub.ox +2H.sup.+( 2)
The redox couple--ferrocene and ferricinium--exhibits a marked difference in UV-VIS absorption spectra. Ferrocene in 95% ethanol is yellow and features two wavelength bands at 325 and 440 nm. Ferricinium solutions exhibit a characteristic dichroism with an absorption peak at 620 nm, i.e. dilute solutions appear blue or green, while more concentrated solutions are blood red. Apparently, ferricinium or its derivatives could be used in spectrophotometric assays involving oxidoreductases if they can be prepared from water-soluble forms of ferrocenerelated compounds. However, ferrocene and its derivatives (except for carboxyl ferrocene) are virtually waterinsoluble.